Biomedical subjects
Publications and source records attributed to J F Watchko.
Jaundice in preterm, as well as full term, infants results from (a) an increased bilirubin load in the hepatocyte, (b) decreased hepatic uptake of bilirubin from the plasma, and/or (c) defective bilirubin conjugation. Hyperbilirubinaemia in preterm infants is more prevalent, more severe, and its course more protracted than in term neonates.
Exchange transfusion and phototherapy remain the staples of intervention for the jaundiced newborn. Clinical management of the jaundiced low birthweight infant is discussed.
The extensor digitorum longus (EDL) and soleus muscles of adult mice were chronically denervated or denervated and allowed to reinnervate. Muscles were evaluated 1, 5, 14, 21, and 52 days after sciaticectomy. In terms of weight loss, myofiber atrophy, degeneration, and fibrosis, the soleus muscle was more affected than the EDL by chronic denervation. Fifty-two days after chronic denervation, the number of molecules of MCK/ng total RNA in both muscles (determined with competitive PCR) decreased, with the soleus muscle being more affected. At that stage, BCK mRNA levels in the denervated soleus were unchanged, but they were increased (>50%) in the EDL. Reinnervation restored MCK transcript accumulation in the EDL, whereas, in the soleus MCK, transcripts exceeded control values by 57%, approaching levels in the reinnervated EDL. Despite restoration of MCK mRNA levels, the number of molecules of BCK mRNA/ng total RNA was four- to fivefold higher in reinnervated versus control muscles, suggesting that the genes encoding the CK mRNAs are not coordinately regulated in adult muscle. The role of denervation induced, fiber type changes in regulating CK mRNA accumulation has been evaluated. Electron microscopic analyses have established that fibrosis is not a factor that determines BCK mRNA levels in the chronically denervated or denervated-reinnervated muscles. CK isozyme analyses support the hypothesis that a greater proportion of BCK mRNA found in 52 day chronically denervated and denervated-reinnervated muscles is produced in myofibers vs. nonmuscle cells than in control muscles.
P-glycoprotein (Pgp), an ATP-dependent plasma membrane efflux pump, is expressed in abundance on the luminal aspect of brain capillary endothelial cells and astrocytes of the blood-brain barrier where it limits the passage of a variety of lipophilic substrates into the central nervous system. This review summarizes current evidence characterizing (1) unconjugated bilirubin as a potential substrate for Pgp and (2) the ontogeny of Pgp expression at the blood-brain barrier and apical brush border epithelium of the gastrointestinal tract, findings that may provide insights regarding the disposition of bilirubin in immature subjects.
BACKGROUND: P-glycoprotein (Pgp) is an ATP-dependent, integral plasma-membrane efflux pump that is constitutively expressed on (i) adult apical brush-border epithelial cells of the intestine, (ii) the bile canalicular face of hepatocytes, and (iii) the brush border epithelium of renal proximal tubules. This Pgp tissue distribution and localization affects the absorption, distribution, metabolism, and excretion of Pgp substrates. Little is known regarding the ontogeny of Pgp expression in these tissues. METHODS: Postnatal expression of Pgp on brush border membranes of small intestine, liver, and kidney as a function of maturity from birth through adulthood was determined using Western immunoblotting and immunohistochemical techniques. Tissue was isolated from FVB mice at four different ages: day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adult (Ad). The relative expression of Pgp protein on Western immunoblots was assessed by scanning densitometry and indexed as a percentage (mean+/-SEM) of the adult levels. RESULTS: On Western immunoblots, Pgp expression was limited at birth (19+/-6% of Ad) and increased significantly with maturation in intestine (ANOVA, P<0.005). In contrast, hepatic (113+/-12% of Ad) and renal (96+/-15% of Ad) Pgp expression were at adult levels at birth. The tissue-specific developmental pattern of Pgp expression was confirmed by immunohistochemistry. CONCLUSIONS: We conclude that Pgp is expressed in a tissue-specific and developmentally regulated fashion and speculate that developmental modulation of intestine-Pgp expression may affect the oral bioavailability of Pgp substrates.
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Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the delta-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74-82, 1999). In this report, we show efficient and long-term delta-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.
Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. The diaphragm (Dia) expresses both M-CK and ScCKmit in abundance. We compared the power and work output of 1) control CK-sufficient (Ctl), 2) M-CK-deficient [M-CK(-/-)], 3) ScCKmit-deficient [ScCKmit(-/-)], and 4) combined M-CK/ScCKmit-deficient null mutant [CK(-/-)] Dia during repetitive isotonic activations to determine the effect of CK phenotype on Dia function. Maximum power was obtained at approximately 0.4 tetanic force in all groups. M-CK(-/-) and ScCKmit(-/-) Dia were able to sustain power and work output at Ctl levels during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load), and the duration of sustained Dia shortening was 67 +/- 4 s in M-CK(-/-), 60 +/- 4 s in ScCKmit(-/-), and 62 +/- 5 s in Ctl Dia. In contrast, CK(-/-) Dia power and work declined acutely and failed to sustain shortening altogether by 40 +/- 6 s. We conclude that Dia power and work output are not absolutely dependent on the presence of either M-CK or ScCKmit, whereas the complete absence of CK acutely impairs Dia shortening capacity during repetitive activation.
The effects of growth hormone (GH) administration and refeeding after chronic undernutrition (UN) were investigated in Fischer 344 male rats aging into senescence (24.5 mo of age) during UN initiated at 12.5 mo of age that produced muscle atrophy and a 50% decrease in body mass. Muscle mass, protein, myosin heavy-chain (MHC) composition and circulating testosterone levels were measured and compared to controls with free access to food. Within 9 wk, refeeding + GH restored body mass to control levels, whereas it was still decreased with refeeding alone. By 24.5 mo of age, refeeding alone restored body mass, while addition of GH resulted in overshoot. UN uniformly decreased mass of the gastrocnemius, extensor digitorum longus, soleus and diaphragm muscles to 50-60% of controls. Refeeding and refeeding + GH restored these losses with some overshoot of gastrocnemius muscle suggesting hypertrophy. UN more than doubled slow Type I MHC composition and approximately halved fast Type IIB and IIX MHC in the deep gastrocnemius muscle while it increased Type IIA MHC in the diaphragm. Refeeding and refeeding + GH reversed these shifts. MHC shifts in the extensor digitorum longus and soleus muscles were not statistically significant, whereas UN increased fast Type IIA MHC followed by decrease with refeeding + GH. UN decreased testosterone levels to nearly zero followed by restoration with refeeding and refeeding + GH. We conclude that the phenotype of mixed-MHC muscles such as the gastrocnemius and diaphragm are most affected by chronic UN, which is reversible with refeeding and refeeding + GH. These alterations were associated with changes in circulating testosterone, which may be a key regulatory element in these processes.
The effects of growth hormone (GH) on diaphragm muscle myosin heavy chain (MHC) composition and mechanical performance were investigated in Fischer 344 male rats aged to senescence (24.5 mo of age). Chronic undernutrition (UN), refeeding (RF), and RF+GH were compared with ad libitum feeding by using a model of UN that produced a 50% decrease in body weight over a 12-mo period. The effect of aging was assessed by comparing MHC composition of ad libitum-fed rats at 12 and 24.5 mo of age. At senescence, significant decreases in slow type I (-23%) and fast type IIA (-31%) MHC had occurred with aging. Conversely, UN over this aging period increased types I (32-73%) and IIA (22-23%) MHC and decreased fast types IIB (32-54%) and IIX (30-31%) MHC. RF and RF+GH reversed these shifts back toward control values. At senescence, maximal specific force, maximal velocity, and specific power capacity were not different across treatment groups. During repetitive isotonic contraction trials, the diaphragms of UN rats maintained power production over time (54% of initial power at 60 s), whereas the power production of diaphragms of ad libitum-fed rats fell to 0% (P < 0.05). In comparison with UN rats, the diaphragms of RF and RF+GH rats produced 23 (not significant) and 11% (P < 0.05) of initial power, respectively, suggesting that RF+GH treatment restored performance characteristics after UN. We conclude that RF+GH can reverse alterations in MHC composition and mechanical performance produced by chronic UN in the aged rat diaphragm.
Previous investigations have shown that ventilatory failure during severe inspiratory resistive loading (IRL) in the 21-day-old infant primate occurs secondary to a decrease in respiratory frequency, that is, central failure. To examine the response of the more immature newborn to IRL, minute ventilation (V'E), arterial blood gases and pH, minute diaphragmatic electromyogram (EMG) activity, peak inspiratory airway pressure, and the centroid frequency (Fc) of the diaphragmatic EMG power spectrum were measured in four unanesthetized tracheotomized 2-day-old monkeys during various levels of IRL, until either 1) ventilatory failure occurred (ventilatory failure run) or 2) normocapnia was sustained for 1 hr (successful trial). During successful trials, minute ventilation, breathing frequency, tidal volume, Fc, and PaCO2 were sustained at baseline levels and an increase in minute EMG activity and peak inspiratory airway pressure were observed. In contrast, during ventilatory failure runs, minute ventilation and tidal volume fell and PaCO2 rose compared to their respective baseline values. Respiratory frequency did not change. The decline in tidal volume occurred despite significant increases in minute diaphragmatic EMG activity and peak inspiratory airway pressure. No shifts in Fc were noted, suggesting that peripheral diaphragmatic fatigue did not occur. We conclude that ventilatory failure during IRL in the 2-day-old monkey is due to the animal's inability to defend tidal volume as opposed to central failure.
Growth hormone (GH) was administered (1 mg/day, i.p., 7.5 months) to male Fischer 344 rats, in conjunction with refeeding (RF) after chronic undernutrition (UN), from middle age (17 months old) to senescence (24.5 months old), during which cardiac myosin heavy chain (MHC) profiles were determined by gel-electrophoresis. At 17 months of age, respective MHC-alpha and -beta composition was 74 and 26% in the right ventricle (RV), and 58 and 42% in the left ventricle (LV), of ad libitum-fed controls. At 24.5 months of age, MHC profiles of controls were shifted toward the MHC-beta isoform in both RV (alpha=53%, beta=47%) and LV (alpha=40%, beta=60%), indicating a significant effect of aging on MHC composition in both ventricles. At 17 months of age, 7.5 months of UN likewise resulted in a shift toward the MHC-beta isoform in both RV (alpha=31%, beta=69%) and LV (alpha=22%, beta=78%) as compared to controls, indicating a significant effect of UN in both ventricles. Continued UN into senescence maintained these altered profiles in both ventricles, at 24.5 months of age (RV: alpha=35%, beta=65%; LV: alpha=24%, beta=76%). RF+GH administered from middle age into senescence restored the MHC composition in both ventricles (RV: alpha=57%, beta=43%; LV: alpha=43%, beta=57%), to that of the controls. RF, alone, likewise reversed ventricular MHC composition toward that of MHC-alpha, but appeared to overcompensate (RV: alpha=67%, beta=33%; LV: alpha=46%, beta=54%), surpassing the control and RF+GH profiles, significantly in the RV. These data suggest that GH is a modulator of restoration of cardiac MHC composition, when RF is administered to counter the effects of chronic UN, in the aging rat heart.
The effects of chronic undernutrition (UN) on respiratory muscle were investigated during UN producing a 50% decrease in body weight over a prolonged period (45 weeks) in Fischer 344 male rats. This model focused on progressive, aging-related changes in myosin heavy chain (MHC) profile over time, in which the confounding effects of early development and late senescence were avoided. With aging toward late adulthood (68 weeks), MHC composition of control diaphragms was shifted, with decreased type I (slow) and IIA MHC, and increased type IIB and IIX (fast) MHC. UN produced a divergence of this profile, with an increase in type I and IIA MHC, and decreased type IIX MHC. UN diaphragms in vitro were more resistant to loss of active force with fatigue, during repetitive contractions. However, passive tension rose disproportionately during fatigue, suggesting increased fatigability. We conclude that the observed changes in diaphragm mechanical function are consistent with the UN-induced shifts in MHC composition; however, the elevated passive tension with fatigue suggests additional UN-induced changes in mechanical properties that are possibly detrimental to respiratory muscle function. The UN-dependent divergence in phenotype and mechanical properties may be amplified by aging-related shifts in muscle MHC composition over time, in the control group.
The myosin heavy chain (MHC) exists as multiple isoforms that are encoded for by a family of genes. The respiratory musculature demonstrates muscle-specific and temporally-dependent changes in MHC isoform expression during maturation. Developmental expression of MHC isoforms correlate well with postnatal changes in actomyosin ATPase activity, specific force generation (P0/CSA), maximum unloaded velocity of shortening (V0) and and fatigue resistance. More specifically, as the expression of MHCneonatal declines and MHC2A, MHC2X, and MHC2B increase, actomyosin ATPase activity, P0/CSA, V0, and muscle fatigability increase. The increase in actomyosin ATPase activity with maturation is partially offset by a postnatal increase in oxidative capacity; however, as fatigue resistance declines with development it is apparent that the energy costs of contraction are not fully matched by an increase in energy production. Developmental transitions in smooth muscle MHC phenotype also occur although their functional importance remains unclear.
Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. We compared the isometric contractile and fatigue properties of 1) control CK-sufficient (Ctl), 2) M-CK-deficient (M-CK[-/-]), and 3) combined M-CK/ScCKmit-deficient null mutant (CK[-/-]) diaphragm (Dia) to determine the effect of the absence of M-CK activity on Dia performance in vitro. Baseline contractile properties were comparable across groups except for specific force, which was approximately 16% lower in CK[-/-] Dia compared with M-CK[-/-] and Ctl Dia. During repetitive activation (40 Hz, (1)/(3) duty cycle), force declined in all three groups. This decline was significantly greater in CK[-/-] Dia compared with Ctl and M-CK[-/-] Dia. The pattern of force decline did not differ between M-CK[-/-] and Ctl Dia. We conclude that Dia isometric muscle function is not absolutely dependent on the presence of M-CK, whereas the complete absence of CK acutely impairs isometric force generation during repetitive activation.
Postnatal transitions in myosin heavy chain (MHC) isoform expression were found to be associated with changes in both isometric and isotonic contractile properties of rat diaphragm muscle (Diam). Expression of MHCneo predominated in neonatal Diam fibers but was usually coexpressed with MHCslow or MHC2A isoforms. Expression of MHCneo disappeared by day 28. Expression of MHC2X and MHC2B emerged at day 14 and increased thereafter. Associated with these MHC transitions in the Diam, maximum isometric tetanic force (Po), maximum shortening velocity, and maximum power output progressively increased during early postnatal development. Maximum power output of the Diam occurred at approximately 40% Po at days 0 and 7 and at approximately 30% Po in older animals. Susceptibility to isometric and isotonic fatigue, defined as a decline in force and power output during repetitive activation, respectively, increased with maturation. Isotonic endurance time, defined as the time for maximum power output to decline to zero, progressively decreased with maturation. In contrast, isometric endurance time, defined as the time for force to decline to 30-40% Po, remained > 300 s until after day 28. We speculate that with the postnatal transition to MHC2X and MHC2B expression energy requirements for contraction increase, especially during isotonic shortening, leading to a greater imbalance between energy supply and demand.
A profile of respiratory complications has been associated with the onset and development of obesity in humans. Similar phenotypes have been routinely demonstrated in genetic animal models of obesity such as the ob mouse (C57BL/6J-Lepob). The objective of the present study was to test the hypothesis that a constellation of respiratory complications are attenuated with leptin (i.e., protein product of the ob gene) replacement. Daily leptin administration during a 6-wk period was conducted to control body weight of mutant ob mice similar to genotypic control groups. During the treatment period, repeated baseline ventilatory measurements were assessed by using whole body plethysmography while quasistatic pressure-volume curves were performed to further explore the role of leptin in improving lung mechanics. Diaphragmatic myosin heavy chain (MHC) isoform phenotype was examined to determine proportional changes in MHC composition. In room air, breathing frequency and minute ventilation were significantly (P < 0.01) different among ob treatment groups, suggesting that leptin opposed the development of a rapid breathing pattern observed in vehicle-treated ob mice. Quasistatic deflation curves indicated that the lung volume of leptin-treated ob mice was significantly (P < 0.05) greater relative to vehicle-treated ob mice at airway pressures between 0 and 30 cmH2O. Diaphragm MHC composition of leptin-treated ob mice was restored significantly (P < 0.05) to resemble the control phenotype. In this genetic mouse model of obesity, the results suggested that respiratory complications associated with the obese phenotype, including rapid breathing pattern at baseline, diminished lung compliance, and abnormal respiratory muscle adaptations, are attenuated with prolonged leptin treatment.
P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean +/- SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 +/- 2.4 nmol/g) compared with (+/+) mice (10.4 +/- 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 +/- 1.4 nmol/g) compared with (+/+) mice (5.3 +/- 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t 1/2 approximately 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.